Use of diazolidinyl urea for anti-clumping of biological samples

ABSTRACT

The present invention provides methods for preventing clumping of cells in microfluidic devices by addition of diazolidinyl urea (DU). DU can be added to samples at the time of collection or can be added to samples post-collection. DU can also be pre-added to sample collection devices.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.:61/385,935 filed Sep. 23, 2010, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to the use of diazolidinyl urea (DU) and itsrelated compounds, e.g., the use of DU to prevent cell clumping inbiological samples.

BACKGROUND OF THE INVENTION

When biological samples including cells or cellular components arecollected and/or processed for testing, these cells or cellularcomponents can clump or stick together. This could be problematic forthe testing down stream, e.g., this may block the flow of fluid throughtesting devices or make sample analysis difficult. In particular,whenever samples are “stressed” either through shipment orpre-processing, they begin to clump up when passing through testingdevices, e.g., microfluidic devices. For example, older samples andsamples from patients with medical conditions accentuate such clumpingeffect even further. In addition, pre-processing of samples, forexample, through density gradients or centrifugation steps activates theclumping effect.

There is a need in the field for methods or reagents useful foraddressing the cell clumping issue in sample collection and processing.

BRIEF SUMMARY OF THE INVENTION

The present invention is based at least in part on the discovery thatdiazolidinyl urea or its derivatives or analogs can be used to preventor reduce cell clumping in biological samples. Accordingly the presentinvention provides methods and devices useful for preventing or reducingcell clumping in biological samples. In some embodiments, the presentinvention provides methods for inhibiting cellular clumping in abiological sample by mixing an effective amount of diazolidinyl urea(DU) with the sample, e.g., where the cellular clumping is reduced atleast 50% in the presence of DU compared to the absence of DU in thesample.

In some embodiments, the effective amount of diazolidinyl urea is atleast 0.01%-3%.

In some embodiments, the method includes mixing an effective amount ofdiazolidinyl urea in a combination with a non-chelating agent basedanti-coagulating agent with the biological sample. In some embodiments,the methods include mixing an effective amount of diazolidinyl urea incombination with acid citrate dextrose (ACD) with the biological sample.

In some embodiments, the diazolidinyl urea is mixed with the biologicalsample before or during biological sample testing.

In some embodiments, the diazolidinyl urea can be in a solid or liquidform in a container, where the biological sample is added to thecontainer.

In some embodiments, the biological sample is selected from the groupconsisting of whole blood, blood serum, plasma and bone marrow.

The present invention also provides a container for collectingbiological samples comprising an effective amount of diazolidinyl ureain combination with a non-chelating agent based anti-coagulating agent.In some embodiments, the volume of the container is from about 0.5 mL toabout 50 mL and the diazolidinyl urea is in an amount from about 0.02grams to about 30 grams. In some embodiments, the non-chelating agentbased anti-coagulating agent in the container is acid citrate dextrose.In some embodiments, the container comprises a biological sample. Insome embodiments, the diazolidinyl urea in the container is in a liquidor solid form.

The present invention also provides a container for collectingbiological samples comprising an effective amount of diazolidinyl ureain an amount of at least about 0.01% to about 3% upon mixing with abiological sample. In some embodiments, the container comprises anon-chelating agent based anti-coagulating agent. In some embodiments,the container comprises an effective amount of acid citrate dextrose. Insome embodiments, the container comprises a biological sample. In someembodiments, the diazolidinyl urea in the container is in a liquid orsolid form.

The present invention also provides a container for collectingbiological samples having a size from about 0.5 mL to about 50 mL anddiazolidinyl urea in an amount from about 0.02 grams to about 30 grams.In some embodiments, the container includes an instruction for using thecontainer to collect a biological sample. In some embodiments, thediazolidinyl urea is in a solid or liquid form.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Example of microfluidic device processed with blood without DU.The white strands are cell clumps.

FIG. 2. Example of microfluidic device processed with blood containingDU (after incubating samples overnight with 2% DU).

FIG. 3. Example of microfluidic device processed with blood without DU.

FIG. 4. Example of microfluidic device processed with blood containingDU (after incubating samples for 30 minutes with 2% DU).

DETAILED DESCRIPTION

The present invention is based at least in part on the discovery thatdiazolidinyl urea or its derivatives or analogs can be used to preventor reduce cell clumping in biological samples. Accordingly the presentinvention provides methods and devices useful for inhibiting, preventingor reducing cell clumping in biological samples.

According to one aspect of the present invention, it provides methodsfor inhibiting cellular clumping in a biological sample by mixing aneffective amount of diazolidinyl urea (DU) or its derivatives or analogswith the sample, e.g., where the cellular clumping is reduced at least50% in the presence of DU compared to the absence of DU in the sample.

According to the present invention, cellular clumping includes anycomplex of two or more cells or cellular components, which includeswithout any limitation any cellular membrane encapsulated entity, e.g.,microsomes, microvesicles, exosomes, mitochondria, etc. In someembodiments, cellular clumping includes two or more cells or cellularcomponents in contact with each other, e.g., in reversible orirreversible contact with each other. In some other embodiments,cellular clumping includes two or more cells or cellular components thataggregate together in close proximity. In some other embodiments,cellular clumping includes two or more cells or cellular components inclose proximity and moving as one group.

In general, cellular clumping can occur before (pre-processing orpre-testing), after or during sample processing or testing. In someinstances, cellular clumping can occur in the presence or absence ofsample processing. In some cases, the amount of cellular clumpingincreases the longer a biological sample is stored, or the more abiological sample is manipulated and/or stressed (for example by beingshipped). In some cases, sample processing and pre-processing canincrease cellular clumping, for example when certain reagents are addedto the sample or when the biological sample is mechanically stressed.For example, mechanical stress may occur due to pipetting, passagethrough density gradients, centrifugation, passage through a syringeand/or passage through a device such as a microchannel or microfluidicdevice.

Sample processing or testing can be performed according to any standardprocedure known in the art. In conjunction with the present invention,biological samples treated according to the present invention can beprocessed for use or testing in a variety of cellular, molecular,diagnostic and clinical assays. Exemplary assays that are contemplatedfor use with the present invention can include immunoassays (such asELISA), cell sorting assays (such as but not limited to FACS), flowcytometry assays, nucleic acid assays (including RNA and DNA detectionand/or isolation), protein assays (including detection and isolation),drug interaction assays, microfluidic assays, rare cell detection orquantitation, or any other variety of sorting, detection or quantitationassays that are presently used or later become available in the art. Anyassay where preventing or inhibiting cellular clumping would provide abenefit is contemplated for use with methods and products of the presentinvention. In some embodiments, the assays are manual or automated. Insome embodiments, the assays include the use of devices, such as but notlimited to microfluidic devices or microchannels.

In some embodiments, cellular clumping is reduced by about 10%, about20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about100%, about 150% or about 200% or more, e.g., caused by the addition ofdiazolidinyl urea. In some embodiments, cellular clumping is reduced byabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9 or about 10 fold or more, e.g., caused by the addition ofdiazolidinyl urea. Any suitable methods or assays known or later becomeavailable can be used to measure cellular clumping in a sample in thepresence or absence of DU. For example, cellular clumping can bemeasured by visually surveying the extent of cellular clumping under amicroscope.

According to the present invention, diazolidinyl urea includesdiazolidinyl urea, and optionally its derivatives and analogs. In someembodiments, diazolidinyl urea includes any DU derivatives or analogsthat are formaldehyde donors, e.g., DMDM hydantoin, imidiazolidinylurea, or quaternium 15. In some embodiments, diazolidinyl urea includesany DU derivative or analogs that is not a preservative or stabilizingagent. In some embodiments, diazolidinyl urea includes substantiallydiazolidinyl urea and one or more other non-active components, e.g.,components that do not materially change the activity of DU. In someembodiments, diazolidinyl urea includes a composition of diazolidinylurea consisting essentially of DU.

In general, the effective amount of diazolidinyl urea is any amount thatreduces cellular clumping in a biological sample. In some embodiments,the effective amount of DU is an amount that provides substantialanti-cellular clumping activity, but insubstantial activity forstabilizing or preserving cells or cellular components in a biologicalsample. In some embodiments, the effective amount of DU is an amountdifferent from what DU is normally used for as preservatives or fixingagent, e.g., less than what DU is normally used for as preservatives orfixing agent. In some other embodiments, the effective amount of DU isan amount that achieves at least about 10%, 20%, 30%, 40% or 50%reduction in cellular clumping in a biological sample. In someembodiments, the effective amount of diazolidinyl urea is at least about0.01% to about 3% in a biological sample. In some embodiments, theeffective amount of diazolidinyl urea is at least about 0.01%, about0.05%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.5%,about 1%, about 1.5%, about 2%, about 2.5% or about 3% in a biologicalsample.

According to the present invention, in some embodiments diazolidinylurea can be used in combination with one or more non-chelating agentbased anti-coagulating agents. For example, DU and one or more (same ordifferent) non-chelating agent based anti-coagulating agent can be addedtogether or separately (sequentially or concurrently) to a biologicalsample. Non-chelating agent based anti-coagulating agents are thoseanti-coagulating agents with chelating affinity less than the chelatingaffinity of EDTA. Methods for determining chelating affinity are wellknown in the field and that are presently used or later become availablein the art can be used. Any non-chelating agent based anti-coagulatingagent is contemplated for use with the present invention. Examples ofnon-chelating agent based anti-coagulating agents can include but arenot limited to acid citrate dextrose (ACD), citrate,citrate-theophylline-adenosine-dipuridamole (CTAD),citrate-pyridoxalphosphate-tris, heparin-β-hydroxy-ethyl-theophylline,polyanethol sulfonate, sodium fluoride, sodium heparin, clot activator,serum separator, thrombin and PPACK (D-phenylalanyl-L-prolyl-L-argininechloromethyl ketone). In some embodiments, the non-chelating agent basedanti-coagulating agent is acid citrate dextrose (ACD), citrate,citrate-theophylline-adenosine-dipuridamole (CTAD),citrate-pyridoxalphosphate-tris, heparin-β-hydroxy-ethyl-theophylline,polyanethol sulfonate, sodium fluoride, sodium heparin, clot activator,serum separator, thrombin or PPACK (D-phenylalanyl-L-prolyl-L-argininechloromethyl ketone). In some embodiments, the non-chelating agent basedanti-coagulating agent is acid citrate dextrose (ACD). In someembodiments, the non-chelating agent based anti-coagulating agent iscitrate (citric acid).

In some embodiments, the combination of diazolidinyl urea and anon-chelating agent based anti-coagulating agent includes one or moreother ingredients, e.g., preservatives, stabilizing agents, fixingagents, antibiotics, etc. In some embodiments, the combination ofdiazolidinyl urea and a non-chelating agent based anti-coagulating agentincludes no other active ingredients, e.g., includes no otheringredients that materially change the activity of DU and non-chelatingagent based anti-coagulating agent. In some embodiments, the combinationof diazolidinyl urea and a non-chelating agent based anti-coagulatingagent does not include a chelating based anti-coagulating agent, e.g.,EDTA or other similar anti-coagulating agent. In some embodiments, thecombination of DU and a non-chelating agent based anti-coagulating agentconsists essentially of DU and a non-chelating agent basedanti-coagulating agent, e.g., acid citrate dextrose or citrate (citricacid).

According to the present invention, a biological sample can include anyunprocessed or processed cell, tissue or human secretion samples. Insome embodiments, a biological sample includes any sample which containscells or cellular components. In some embodiments, a biological sampleis whole blood, blood serum, plasma, bone marrow, saliva, aqueoushumour, vitreous humour, bile, breast milk, cerebrospinal fluid, cerumen(earwax), endolymph and perilymph, female ejaculate, gastric juice,mucus (including nasal drainage and phlegm), peritoneal fluid, pleuralfluid, sebum (skin oil), semen, sweat, tears, vaginal excretion, vomitor urine. In some other embodiments, the biological sample is wholeblood, blood serum, plasma or bone marrow.

According to the present invention, diazolidinyl urea and optionally oneor more non-chelating agent based anti-coagulating agent can be added tothe biological sample at the time of collection, during pre-processingor pre-testing steps or at the time of processing or testing. In someembodiments, diazolidinyl urea and optionally one or more non-chelatingagent based anti-coagulating agent is added to a biological sample atthe time of collection. In some embodiments, diazolidinyl urea andoptionally one or more non-chelating agent based anti-coagulating agentis added to samples upon receipt or at any stage of pre-processing orpre-testing. In some embodiments, the diazolidinyl urea and optionallyone or more non-chelating agent based anti-coagulating agent is mixedwith the biological sample before sample processing or testing. In someembodiments, the diazolidinyl urea and optionally one or morenon-chelating agent based anti-coagulating agent is mixed with thebiological sample during biological sample processing or testing. Insome embodiments, diazolidinyl urea and optionally one or morenon-chelating agent based anti-coagulating agent is added to the samplesbefore the samples are loaded into an assay device. In some embodiments,diazolidinyl urea and optionally one or more non-chelating agent basedanti-coagulating agent is added to the samples before the samples areloaded onto a testing device, e.g., microfluidic device or microchannel.

In some embodiments, diazolidinyl urea and optionally one or morenon-chelating agent based anti-coagulating agent can be included orbuilt-in in a sample collection device, e.g., be present in the deviceprior to the addition of biological sample. In some embodiments, thebiological samples can be collected directly into a diazolidinyl ureacontaining device and optionally the device may contain one or morenon-chelating agent based anti-coagulating agent.

In some embodiments, biological samples are collected without additionof diazolidinyl urea. According to the present invention, whenbiological samples are collected without diazolidinyl, the diazolidinylurea can be subsequently added to the biological sample. In someembodiments, diazolidinyl urea is added after biological samplecollection. In some embodiments, diazolidinyl urea is added to samplesduring any pre-processing or pre-testing steps.

The diazolidinyl urea of the present invention can be in a solid orliquid form. In some embodiments the diazolidinyl urea is dissolved toform an about 4%, about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90% or more weight/volume (w/v)mixture. In general, diazolidinyl urea can be dissolved in any solutionthat would be appropriate for the intended sample processing or testing.One skilled in the art can readily determine suitable solutions fordissolving DU and optionally one or more non-chelating agent basedanti-coagulating agent. In some embodiments the diazolidinyl urea isdissolved in PBS. In some embodiments the diazolidinyl urea is dissolvedin PBS to form an 80% (w/v) mixture.

In some embodiments, the diazolidinyl urea is dissolved in a solutioncontaining a non-chelating agent based anti-coagulating agent. In someembodiments, the diazolidinyl urea is dissolved in a solution containingACD. In some embodiments, the ACD solution contains about 10 grams toabout 25 grams of citrate (citric acid), about 10 grams to about 30grams of dextrose and about 2 to about 10 grams of sodium per 1000 mLvolume. In some embodiments, the ACD solution contains about 12 grams toabout 25 grams of citrate (citric acid), about 13 grams to about 26grams of dextrose and about 3 to about 6 grams of sodium per 1000 mLvolume. In some embodiments, the ACD solution contains about 20 grams toabout 23 grams of citrate (citric acid), about 23 grams to about 26grams of dextrose and about 4 to about 6 grams of sodium per 1000 mLvolume. In some embodiments, the ACD solution contains about 12 grams toabout 14 grams of citrate (citric acid), about 13 grams to about 16grams of dextrose and about 2 to about 4 grams of sodium per 1000 mLvolume. The non-chelating agent based anti-coagulating agent solutioncan be made up and used according to standard methods known in the artfor such solutions. Non-chelating agent based anti-coagulating agentscan also be made up according to standard procedures known in the art.For example, ACD can be made up according to any standard formulation,sometimes referred to as Solution A and Solution B. ACD can be made upfor example according to the formulations listed in Tables 1 and 2.

TABLE 1 ACD Formulations Formulation 1* Formulation 2* citrate (citricacid C₆H₈O₇)  7.3 g  4.4 g sodium citrate (dihydrate) 22.0 g 13.2 gdextrose (monohydrate; C₆H₁₂O₆*H₂O) 24.5 g 14.7 g *Per 1000 mL volume.

TABLE 2 ACD Formulations Formulation 3* Formulation 4* citrate (citricacid C₆H₈O₇)  8.0 g  4.8 g trisodium citrate 22.0 g 13.2 g dextrose 24.5g 14.7 g *Per 1000 mL volume.

In some other embodiments, diazolidinyl urea is dissolved in a solutioncontaining the non-chelating agent based anti-coagulating agent citrate(citric acid). In some embodiments, the citrate solution contains about10 grams to about 25 grams of citrate (citric acid) per 1000 mL volume.In some embodiments, the citrate solution contains about 12 grams toabout 25 grams of citrate per 1000 mL volume. In some embodiments, thecitrate solution contains about 20 grams to about 23 grams of citrate(citric acid) per 1000 mL volume. In some embodiments, the citratesolution contains about 12 grams to about 14 grams of citrate (citricacid) per 1000 mL volume.

The ACD solution can be mixed with diazolidinyl urea (DU) before orafter addition to the biological sample. In some embodiments, about 0.5mL to about 5 mL of ACD/DU is added to the biological sample. In someembodiments, the ACD/DU solution is mixed at about 1 part ACD/DU toabout 10 parts biological sample. In some embodiments, the ACD/DUsolution is mixed at about 1 part ACD/DU to about 8 parts biologicalsample. In some embodiments, the ACD/DU solution is mixed at about 1part ACD/DU to about 4 parts biological sample. In some embodiments, theACD/DU is mixed with the biological sample at a ratio of about 1 toabout 8 in an about 10 mL tube.

The citrate solution can be mixed with diazolidinyl urea (DU) before orafter addition to the biological sample. In some embodiments, about 0.5mL to about 5 mL of citrate/DU is added to the biological sample. Insome embodiments, the citrate/DU solution is mixed at about 1 partcitrate/DU to about 10 parts biological sample. In some embodiments, thecitrate/DU solution is mixed at about 1 part citrate/DU to about 8 partsbiological sample. In some embodiments, the citrate/DU solution is mixedat about 1 part citrate/DU to about 4 parts biological sample. In someembodiments, the citrate/DU is mixed with the biological sample at aratio of about 1 to about 8 in an about 10 mL tube.

According to another aspect of the present invention, it provides samplecontainers with built-in DU and optionally one or more non-chelatingagent based anti-coagulating agent. In some embodiments, the samplecontainer of the present invention contains DU and one or morenon-chelating agent based anti-coagulating agent, e.g., in a solid orliquid form. In some embodiments, the sample container of the presentinvention contains DU and acid citrate dextrose. In some embodiments,the sample container of the present invention contains DU and citrate(citric acid). In some embodiments, the diazolidinyl urea is alreadypresent in a container when the biological sample is added to thecontainer. In some embodiments, the container includes a compositionconsisting essentially of diazolidinyl urea in combination with anon-chelating agent based anti-coagulating agent. In some embodiments,the container includes a composition, e.g., a liquid compositionconsisting essentially of diazolidinyl urea in combination with acidcitrate dextrose. In some embodiments, the container includes acomposition, e.g., a liquid composition consisting essentially ofdiazolidinyl urea in combination with citrate (citric acid). In someembodiments, the container does not contain a chelating agent basedanti-coagulating agent. In some embodiments, the container does notcontain EDTA. In some embodiments, the sample container of the presentinvention contains a liquid solution comprising or consistingessentially of DU and optionally one or more non-chelating agent basedanti-coagulating agent.

In some embodiments, the sample container of the present invention has asize from about 0.1 mL to about 100 mL, from about 0.5 mL to about 50mL, from about 1 mL to about 10 mL. In some embodiments, the samplecontainer of the present invention has a size from about 0.5 mL to about50 mL and contains DU at a concentration of about 4%, about 5%, about10%, about 20%, about 30%, about 40%, about 50% or about 60% in a liquidsolution in the absence of the biological sample.

In some other embodiments, the sample container of the present inventioncontains an effective amount of DU, in an amount of at least about0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 1.5%, about2%, about 2.5%, about 3%, about 3.5%, about 4%, about 5%, about 6% orabout 10% or more upon mixing with a biological sample.

In some other embodiments, the sample container of the present inventionhas a size from about 0.5 mL to about 50 mL and contains diazolidinylurea in an amount from about 0.02 grams to about 30 grams, e.g., in aliquid or solid form. In some embodiments, the sample container has asize from about 2 mL to about 40 mL and contains diazolidinyl urea in anamount from about 0.08 grams to about 24 grams. In some embodiments, thesample container has a size from about 5 mL to about 30 mL and containsdiazolidinyl urea in an amount from about 0.2 grams to about 18 grams.In some embodiments, the sample container has a size from about 10 mL toabout 20 mL and contains diazolidinyl urea in an amount from about 0.4grams to about 12 grams. In some embodiments, the sample container has asize of about 10 mL and contains diazolidinyl urea in an amount fromabout 0.4 grams to about 6 grams.

In some embodiments, the sample container can also optionally include aninstruction for using the sample container of the present invention tocollect or receive a biological sample. According to the presentinvention, diazolidinyl urea individually or collectively with one ormore non-chelating agent based anti-coagulating agent can be incubatedwith a biological sample for 1 minute, 5 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 4hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96hours or more.

According to the present invention, the sample container of the presentinvention can have built-in DU and optionally one or more non-chelatingagent based anti-coagulating agent either directly inside the samplecontainer or provided in a separate container or device. Accordingly,the present invention provides a sample kit including a sample containerand a reagent container, e.g., DU container. In some embodiments, thesample kit includes just the sample container containing DU and anon-chelating agent based anti-coagulating agent inside the samplecontainer. In some embodiments, the sample kit includes the samplecontainer containing one or more non-chelating agent basedanti-coagulating agent and the reagent container containing DU. In someembodiments, the sample kit includes the sample container and thereagent container containing DU and one or more non-chelating agentbased anti-coagulating agent.

In some embodiments, the reagent container is a syringe. For example,syringes containing concentrated diazolidinyl urea can be included withthe sample kit. Once biological samples are collected in the samplecontainer, diazolidinyl urea can then be subsequently added to thebiological sample in the sample container. In some embodiments, thediazolidinyl urea can be injected into the sample container prior to orafter addition of the biological sample. In some embodiments, theconcentration of diazolidinyl urea in the syringe is from about 4% toabout 60%, e.g., for a sample container in a size from about 0.5 mL toabout 50 mL. In some embodiments, the concentration of diazolidinyl ureain the syringe is from about 10% to about 50%. In some embodiments, theconcentration of diazolidinyl urea in the syringe is from about 15% toabout 40% in the absence of the biological sample. In some embodiments,the concentration of diazolidinyl urea in the syringe is from about 20%to about 30%. In some embodiments, the concentration of diazolidinylurea in the syringe is from about 0.1% to about 30%. In someembodiments, the concentration of diazolidinyl urea in the syringe isfrom about 0.08% to about 24%. For all the concentrations provided inthis paragraph, the corresponding volume of the sample container is fromabout 0.5 mL to about 50 mL.

In some embodiments, the diazolidinyl urea of the present invention isin a solid, semi-solid or liquid form. For example, if the diazolidinylurea is in a solid form in a sample container or a reagent container, aliquid solution could be added to the diazolidinyl urea prior toaddition or exposure of diazolidinyl urea to a biological sample.Methods for diluting solids into solutions are well known in the field.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Treatment of Biological Samples With Diazolidinyl Urea

Diazolidinyl urea has an empirical formula of C₈H₁₄N₄₀₇. Its CAS numberis [78491-02-08].

In this experiment, DU was dissolved in PBS to make an 80% (w/v) aqueousconcentrated solution. The concentrated solution was then added tobiological samples in such a way as to yield a final workingconcentration of 2%. Biological samples were gently mixed with DUsolution to ensure equal distribution of DU, and the resulting mixturewas incubated at room temperature for at least 30 minutes.

Samples were then processed according to pre-determined protocols.

FIG. 1 provides an example of samples without being treated with DU andprocessed through a microfluidic device. The white strands are cellclumps.

FIG. 2 provides an example of samples being treated with DU andprocessed through a microfluidic device (for this particular experiment,the sample and DU were incubated overnight).

FIG. 3 provides another example of samples without being treated with DUand processed through a microfluidic device.

FIG. 4 provides another example of samples being treated with DU andprocessed through a microfluidic device (for this particular experiment,the sample and DU were incubated for 30 minutes.

These experiments demonstrate that DU treatment provides greatimprovement for reducing cellular clumping.

All publications discussed and cited herein are incorporated herein byreference in their entireties. It is understood that the disclosedinvention is not limited to the particular methodology, protocols andmaterials described as these can vary. It is also understood that theterminology used herein is for the purposes of describing particularembodiments only and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the appended claims.

1.-7. (canceled)
 8. A container for collecting biological samplescomprising an effective amount of diazolidinyl urea sufficient forinhibiting cellular aggregation in combination with a non-chelatingagent based anti-coagulating agent, wherein the effective amount ofdiazolidinyl urea is an amount that provides substantialanti-cellular-aggregation activity but insubstantial activity forpreserving cells in a biological sample upon mixing with the biologicalsample
 9. The container of claim 8, wherein the volume of the containeris from about 0.5 mL to about 50 mL, and wherein the diazolidinyl ureais at a concentration of about 4% to about 60% in the absence of thebiological sample.
 10. The container of claim 8, wherein thenon-chelating agent based anti-coagulating agent is acid citratedextrose.
 11. The container of claim 8, further comprising a biologicalsample.
 12. The container of claim 8, wherein the diazolidinyl urea isin a solid or liquid form.
 13. A container for collecting biologicalsamples comprising an effective amount of diazolidinyl urea sufficientfor inhibiting cellular aggregation, wherein the effective amount ofdiazolidinyl urea is an amount that provides substantialanti-cellular-aggregation activity but insubstantial activity forpreserving cells in a biological sample upon mixing with the biologicalsample.
 14. The container of claim 13, further comprising anon-chelating agent based anti-coagulating agent.
 15. The container ofclaim 13, further comprising an effective amount of acid citratedextrose.
 16. The container of claim 13, further comprising a biologicalsample.
 17. The container of claim 13, wherein the diazolidinyl urea isin a solid or liquid form.
 18. A container for collecting biologicalsamples having a size from about 0.5 mL to about 50 mL and diazolidinylurea in an amount from about 0.02 grams to about 30 grams, wherein theamount of diazolidinyl urea is an amount that provides substantialanti-cellular-aggregation activity but insubstantial activity forpreserving cells in a biological sample upon mixing with the biologicalsample.
 19. The container of claim 8, further comprising an instructionfor using the container to collect a biological sample.
 20. Thecontainer of claim 18, wherein the diazolidinyl urea is in a solid orliquid form.
 21. The container of claim 19 wherein the instructioncomprises using diazolidinyl urea in the amount of about 0.01% to about3% upon mixing with the biological sample.
 22. The container of claim 19wherein the instruction comprises using diazolidinyl urea in the amountof about 0.1% to about 2.5% upon mixing with the biological sample. 23.The container of claim 19 wherein the instruction comprises usingdiazolidinyl urea in the amount of about 0.2% to about 2% upon mixingwith the biological sample.